RESUMO
Acute lymphoblastic leukemias arising from the malignant transformation of B-cell precursors (BCP-ALLs) are protected against chemotherapy by both intrinsic factors as well as by interactions with bone marrow stromal cells. Galectin-1 and Galectin-3 are lectins with overlapping specificity for binding polyLacNAc glycans. Both are expressed by bone marrow stromal cells and by hematopoietic cells but show different patterns of expression, with Galectin-3 dynamically regulated by extrinsic factors such as chemotherapy. In a comparison of Galectin-1 x Galectin-3 double null mutant to wild-type murine BCP-ALL cells, we found reduced migration, inhibition of proliferation, and increased sensitivity to drug treatment in the double knockout cells. Plant-derived carbohydrates GM-CT-01 and GR-MD-02 were used to inhibit extracellular Galectin-1/-3 binding to BCP-ALL cells in co-culture with stromal cells. Treatment with these compounds attenuated migration of the BCP-ALL cells to stromal cells and sensitized human BCP-ALL cells to vincristine and the targeted tyrosine kinase inhibitor nilotinib. Because N-glycan sialylation catalyzed by the enzyme ST6Gal1 can regulate Galectin cell-surface binding, we also compared the ability of BCP-ALL wild-type and ST6Gal1 knockdown cells to resist vincristine treatment when they were co-cultured with Galectin-1 or Galectin-3 knockout stromal cells. Consistent with previous results, stromal Galectin-3 was important for maintaining BCP-ALL fitness during chemotherapy exposure. In contrast, stromal Galectin-1 did not significantly contribute to drug resistance, and there was no clear effect of ST6Gal1-catalysed N-glycan sialylation. Taken together, our results indicate a complicated joint contribution of Galectin-1 and Galectin-3 to BCP-ALL survival, with different roles for endogenous and stromal produced Galectins. These data indicate it will be important to efficiently block both extracellular and intracellular Galectin-1 and Galectin-3 with the goal of reducing BCP-ALL persistence in the protective bone marrow niche during chemotherapy.
Assuntos
Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Camundongos , Animais , Galectina 1/genética , Galectina 1/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Vincristina , Galectinas/metabolismo , Polissacarídeos/metabolismoRESUMO
Environmentally-mediated drug resistance in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) significantly contributes to relapse. Stromal cells in the bone marrow environment protect leukemia cells by secretion of chemokines as cues for BCP-ALL migration towards, and adhesion to, stroma. Stromal cells and BCP-ALL cells communicate through stromal galectin-3. Here, we investigated the significance of stromal galectin-3 to BCP-ALL cells. We used CRISPR/Cas9 genome editing to ablate galectin-3 in stromal cells and found that galectin-3 is dispensable for steady-state BCP-ALL proliferation and viability. However, efficient leukemia migration and adhesion to stromal cells are significantly dependent on stromal galectin-3. Importantly, the loss of stromal galectin-3 production sensitized BCP-ALL cells to conventional chemotherapy. We therefore tested novel carbohydrate-based small molecule compounds (Cpd14 and Cpd17) with high specificity for galectin-3. Consistent with results obtained using galectin-3-knockout stromal cells, treatment of stromal-BCP-ALL co-cultures inhibited BCP-ALL migration and adhesion. Moreover, these compounds induced anti-leukemic responses in BCP-ALL cells, including a dose-dependent reduction of viability and proliferation, the induction of apoptosis and, importantly, the inhibition of drug resistance. Collectively, these findings indicate galectin-3 regulates BCP-ALL cell responses to chemotherapy through the interactions between leukemia cells and the stroma, and show that a combination of galectin-3 inhibition with conventional drugs can sensitize the leukemia cells to chemotherapy.
Assuntos
Galectina 3/antagonistas & inibidores , Galectina 3/metabolismo , Células-Tronco Mesenquimais/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Galectina 3/genética , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Vincristina/farmacologiaRESUMO
In this report, we used liquid chromatography-mass spectrometry and Western blotting to analyze the content and structure of glycosaminoglycans, glycolipids and selected proteins to compare differences between patient-matched normal and cancerous lung tissues obtained from lung cancer patients. The cancer tissue samples contained over twice as much chondroitin sulfate (CS)/dermatan sulfate (DS) as did the normal tissue samples, while the amount of heparan sulfate (HS) and hyaluronan (HA) in normal and cancer tissues were not significantly different. In HS, several minor disaccharide components, including NS6S, NS2S and 2S were significantly lower in cancer tissues, while the levels of major disaccharides, TriS, NS and 0S disaccharides were not significantly different in normal and cancer tissues. In regards to CS/DS, the level of 4S disaccharide (the major component of CS-type A and DS) decreased and the level of 6S disaccharide (the major component of CS- type C) increased in cancer tissues. We also compared the content and structure of GAGs in lung tissues from smoking and non-smoking patients. Analysis of the glycolipids showed all lipids present in these lung tissues, with the exception of sphingomyelin were higher in cancer tissues than in normal tissues. Western analysis showed that syndecan 1 and 2 proteoglycans displayed much higher expression in cancer tissue/biopsy samples. This investigation begins to provide an understanding of patho-physiological roles on glycosaminoglycans and glycolipids and might be useful in identifying potential biomarkers in lung cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/análogos & derivados , Heparitina Sulfato/metabolismo , Ácido Hialurônico/metabolismo , Neoplasias Pulmonares/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/isolamento & purificação , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Sulfatos de Condroitina/química , Sulfatos de Condroitina/isolamento & purificação , Cromatografia Líquida , Dermatan Sulfato/química , Dermatan Sulfato/isolamento & purificação , Dermatan Sulfato/metabolismo , Dissacarídeos/química , Dissacarídeos/isolamento & purificação , Dissacarídeos/metabolismo , Feminino , Glipicanas/química , Glipicanas/isolamento & purificação , Glipicanas/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/isolamento & purificação , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/isolamento & purificação , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fumar/metabolismo , Sindecana-1/química , Sindecana-1/isolamento & purificação , Sindecana-1/metabolismo , Espectrometria de Massas em TandemRESUMO
We present data that hyaluronan (HA) polysaccharides, about 14-86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors.
Assuntos
alfa-Globulinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Sulfatos de Condroitina/metabolismo , Ácido Hialurônico/metabolismo , alfa-Globulinas/genética , Animais , Moléculas de Adesão Celular/genética , Sulfatos de Condroitina/genética , Ácido Hialurônico/genética , CamundongosRESUMO
Decorin proteoglycan is comprised of a core protein containing a single O-linked dermatan sulfate/chondroitin sulfate glycosaminoglycan (GAG) chain. Although the sequence of the decorin core protein is determined by the gene encoding its structure, the structure of its GAG chain is determined in the Golgi. The recent application of modern MS to bikunin, a far simpler chondroitin sulfate proteoglycans, suggests that it has a single or small number of defined sequences. On this basis, a similar approach to sequence the decorin of porcine skin much larger and more structurally complex dermatan sulfate/chondroitin sulfate GAG chain was undertaken. This approach resulted in information on the consistency/variability of its linkage region at the reducing end of the GAG chain, its iduronic acid-rich domain, glucuronic acid-rich domain, and non-reducing end. A general motif for the porcine skin decorin GAG chain was established. A single small decorin GAG chain was sequenced using MS/MS analysis. The data obtained in the study suggest that the decorin GAG chain has a small or a limited number of sequences.
Assuntos
Decorina/química , Glicosaminoglicanos/química , Motivos de Aminoácidos , Animais , Sulfatos de Condroitina/química , Cromatografia/métodos , Biologia Computacional/métodos , Dermatan Sulfato/química , Dissacarídeos/química , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Estrutura Terciária de Proteína , Pele/metabolismo , Suínos , Espectrometria de Massas em Tandem/métodosRESUMO
Placental malaria is a serious problem in sub-Saharan Africa. Young women are particular susceptible to contracting this form of malaria during their first or second pregnancy despite previously acquired immunity from past infections. Placental malaria is caused by Plasmodium falciparum parasites expressing VAR2CSA on the erythrocyte surface. This protein adheres to a low-sulfated chondroitin sulfate-A found in placental tissue causing great harm to both mother and developing fetus. In rare cases, the localization of infected erythrocytes to the placenta can even result in the vertical transmission of malaria. In an effort to better understand this infection, chondroitin sulfate was isolated from the cotyledon part of the placenta, which should be accessible for parasite adhesion, as well as two non-accessible parts of the placenta to serve as controls. The placental chondroitin sulfate structures and their VAR2CSA binding were characterized. All portions of human placenta contained sufficient amounts of the appropriate low-sulfated chondroitin sulfate-A to display high-affinity binding to a recombinant truncated VAR2CSA construct, as determined using surface plasmon resonance. The cotyledon is the only placental tissue accessible to parasites in the bloodstream, suggesting it is the primary receptor for parasite infected red blood cells.
Assuntos
Antígenos de Protozoários/metabolismo , Eritrócitos/metabolismo , Glicosaminoglicanos/metabolismo , Placenta/química , Sítios de Ligação , Sulfatos de Condroitina/química , Eritrócitos/parasitologia , Feminino , Glicosaminoglicanos/química , Glicosaminoglicanos/isolamento & purificação , Humanos , Peso Molecular , Gravidez , Ressonância de Plasmônio de SuperfícieRESUMO
Mitogen-activated protein kinases (MAPKs) cascades play important roles in cell proliferation, death, and differentiation in response to external stimuli. However, the precise role of MAPKs in platycodin D (PD)-induced cytotoxicity remains unclear. In this study, we investigated the anticancer effect of PD and its underlying mechanism on AGS human gastric cancer cells. PD significantly inhibited cell proliferation and induced anoikis, which is a form of apoptosis in which cells detach from the substrate. It showed phosphatidylserine externalization, DNA fragmentation, increase of sub-G1 phase, and activation of caspases in a dose- and time-dependent manner. This apoptosis has been associated with the extrinsic pathway via Fas-L and the intrinsic pathway via mitochondrial Bcl-2 family members. Moreover, PD led to the phosphorylation of stresses-activated protein kinases such as JNK and p38, followed by the activation of AP-1. However, pretreatment with SB203580 (a p38 specific inhibitor) suppressed PD-induced p38 and AP-1 activation, and subsequently attenuated the PD-induced apoptosis in AGS cells. These results suggest that p38 activation is responsible for PD-induced apoptosis in AGS cells and PD might be useful for the development as the anticancer agent of gastric cancer.
Assuntos
Apoptose/efeitos dos fármacos , Saponinas/administração & dosagem , Neoplasias Gástricas , Triterpenos/administração & dosagem , Proteínas Quinases p38 Ativadas por Mitógeno , Anoikis/efeitos dos fármacos , Antineoplásicos/administração & dosagem , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA , Humanos , Imidazóis/farmacologia , Mitocôndrias/metabolismo , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We synthesized a diosgenyl saponin bearing a unique disaccharide from the natural product ß-hederin, together with twelve glycosylated derivatives and determined their cytotoxicity against five different human cancer cell lines. Most of them showed weak cytotoxicity, with the exception of compound , diosgenyl α-L-rhamnopyranosyl-(1â2)-[α-L-arabinopyranosyl-(1â4)]-α-L-arabinopyranoside, which exhibited strong cytotoxicity against A549 cells. The cytotoxicity of was associated with apoptotic cell death, which was characterized by morphological changes, chromatin condensation, DNA fragmentation, and phosphatidylserine externalization. Compound 20 induced apoptosis of A549 cells through a caspase-8-mediated extrinsic pathway and a caspase-9-mediated intrinsic pathway. In addition, phosphorylation of JNK increased but the phosphorylation of ERK decreased after treatment with 20. These results provide a basic mechanism for the anticancer activity of 20.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Saponinas/química , Saponinas/farmacologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Antineoplásicos/síntese química , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Fragmentação do DNA/efeitos dos fármacos , Glicosilação , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/síntese química , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Saponinas/síntese químicaRESUMO
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with mixed-lineage leukemia gene rearrangement (MLL-r) is a poor-prognosis subtype for which additional therapeutic targets are urgently needed. Currently no multi-omics data set for primary MLL r patient cells exists that integrates transcriptomics, proteomics and glycomics to gain an inclusive picture of theranostic targets. Methods: We have integrated transcriptomics, proteomics and glycomics to i) obtain the first inclusive picture of primary patient BCP-ALL cells and identify molecular signatures that distinguish leukemic from normal precursor B-cells and ii) better understand the benefits and limitations of the applied technologies to deliver deep molecular sequence data across major cellular biopolymers. Results: MLL-r cells feature an extensive remodeling of their glycocalyx, with increased levels of Core 2-type O-glycans and complex N-glycans as well as significant changes in sialylation and fucosylation. Notably, glycosaminoglycan remodeling from chondroitin sulfate to heparan sulfate was observed. A survival screen, to determine if glycan remodeling enzymes are redundant, identified MGAT1 and NGLY1, essential components of the N-glycosylation/degradation pathway, as highly relevant within this in vitro screening. OGT and OGA, unique enzymes that regulate intracellular O-GlcNAcylation, were also indispensable. Transcriptomics and proteomics further identified Fes and GALNT7-mediated glycosylation as possible therapeutic targets. While there is overall good correlation between transcriptomics and proteomics data, we demonstrate that a systematic combined multi-omics approach delivers important diagnostic information that is missed when applying a single omics technology. Conclusions: Apart from confirming well-known MLL-r BCP-ALL glycoprotein markers, our integrated multi-omics workflow discovered previously unidentified diagnostic/therapeutic protein targets.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Linhagem Celular Tumoral , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica/genética , Rearranjo Gênico/genética , Glicômica/métodos , Histona-Lisina N-Metiltransferase/genética , Humanos , Leucemia Aguda Bifenotípica/genética , Leucemia Aguda Bifenotípica/metabolismo , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteômica/métodos , Transcriptoma/genéticaRESUMO
Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, is a novel glycosaminoglycan, consisting primarily of the repeating disaccharide structure alpha-D-N-acetylglucosaminyl (1 --> 4) 2-sulfoiduronic acid. AS shows anti-tumor activity in vitro and in vivo. Despite this activity, AS is only weakly cytotoxic towards cancer cells. We examine the interactions between AS and cell-surface proteins in an effort to explain this anti-tumor activity. Using flow cytometry and affinity column chromatography, we confirm that AS has strong affinity to specific cell-surface proteins including nucleolin (NL) in A549 human lung adenocarcinomas. Surprisingly, we found the translocation of NL from nucleus to cytoplasm under the stimulation of AS (100 microg/ml) in vitro. Also, as NL exits the nucleus, the levels of growth factors such as bFGF and signaling cascade proteins, such as p38, p53, and pERK, are altered. These results suggest that the communication between AS and NL plays a critical role on signal transduction in tumor inhibition.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Glicosaminoglicanos/farmacologia , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Ligação Competitiva , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia de Afinidade , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Citometria de Fluxo , Glicosaminoglicanos/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Caramujos/química , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , NucleolinaRESUMO
Human ceruloplasmin, a copper binding alpha(2)-glycoprotein, was purified by a single-step procedure using acharan sulfate affinity chromatography. Acharan sulfate was immobilized to amine-functionalized agarose matrix through carboxylic acids. Ceruloplasmin in human plasma was obtained from 0.4 M NaCl salt elution and characterized by SDS-PAGE (132 and 125 kDa), isoelectric focusing (pI 4.6), Western blotting, and MALDI-TOF-MS peptide mass fingerprinting. Ceruloplasmin was purified 106 fold with a specific oxidase activity of 0.53 U/mg protein.
Assuntos
Ceruloplasmina/isolamento & purificação , Cromatografia de Afinidade/métodos , Glicosaminoglicanos/química , Western Blotting , Ceruloplasmina/química , Eletroforese em Gel de Poliacrilamida , Humanos , Focalização Isoelétrica , Peso Molecular , Mapeamento de Peptídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cimiside E was isolated from the Cimicifuga heracleifolia Komarov extract, which has been previously demonstrated to possess apoptotic action on gastric cancer cells. The IC(50) value of cimiside E on gastric cancer cells for 24 h was 14.58 microM. The mechanism of apoptosis was further elucidated through western blot, RT-PCR, morphology, Annexin V-FITC/PI staining and cell cycle analysis. Cell cycle arrest was induced by cimiside E in S phase at a lower concentration (30 microM) and G2/M phase at higher concentrations (60 and 90 microM). Cimiside E mediated apoptosis through the induction of the caspase cascade for both the extrinsic and intrinsic pathways. These findings suggest that cimiside E may be an effective chemopreventive agent against cancer.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Cimicifuga/química , Fragmentação do DNA/efeitos dos fármacos , Saponinas/farmacologia , Neoplasias Gástricas/patologia , Triterpenos/farmacologia , Anexina A5/metabolismo , Antineoplásicos Fitogênicos/isolamento & purificação , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína Ligante Fas/metabolismo , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saponinas/isolamento & purificação , Triterpenos/isolamento & purificação , Receptor fas/metabolismoRESUMO
Precursor B acute lymphoblastic leukemias (pre-B ALLs) abnormally express a specific glycan structure, 9-O-acetylated sialic acid (9-O-Ac-Sia), on their cell surface, but glycoproteins that carry this modification have not been identified. Using three different lectins that specifically recognize this structure, we establish that nucleolin (NCL), a protein implicated in cancer, contains 9-O-Ac-Sia. Surprisingly, antibodies against the glycolipid 9-O-Ac-Sia GD3 also detected 9-O-Ac-Sia NCL. NCL is present on the surface of pre-B ALL cells as a sialoglycoprotein that is partly 9-O-acetylated and conversely, 9-O-Ac-Sia-containing structures other than NCL are present on these cells as well. Interestingly, NCL and the 9-O-Ac-Sia signal had less co-localization on normal pre-B cells. We also investigated regulation of NCL on the cell surface and found that sialidase treatment increased the percentage of cells positive for cell surface NCL, suggesting that sialylation of NCL promotes internalization. Treatment of pre-B ALL cells with the chemotherapy drug vincristine also increased the percentage of cells with surface NCL and correlated with increased 9-O-Ac-Sia expression. All tested leukemia cells including primary samples expressed NCL, suggesting it as a possible therapeutic target. We confirmed this by showing inhibition of cell proliferation in some pre-B ALLs by exposure to a NCL-specific aptamer AS1411.
Assuntos
Proteínas de Membrana/análise , Fosfoproteínas/análise , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Precursoras de Linfócitos B/química , Proteínas de Ligação a RNA/análise , Sialoglicoproteínas/análise , Células Cultivadas , Humanos , NucleolinaRESUMO
BACKGROUND: Drug resistance of B-cell precursor acute lymphoblastic leukemia (BP-ALL) cells is conferred by both intrinsic and extrinsic factors, which could be targeted to promote chemo-sensitization. Our previous studies showed that Galectin-3, a lectin that clusters galactose-modified glycoproteins and that has both an intracellular and extracellular location, protects different subtypes of BP-ALL cells against chemotherapy. Galectin-1 is related to Galectin-3 and its expression was previously reported to be restricted to the MLL subtype of BP-ALL. METHODS AND RESULTS: Here, we report that Galectin-1 is expressed at different levels in and on different subclasses of BP-ALLs. Bone marrow plasma also contains high levels of Galectin-1. PTX008 is an allosteric inhibitor which inhibits Galectin-1 but not Galectin-3-mediated agglutination. The compound reduces migration of BP-ALL cells to CXCL12 and OP9 stromal cells and inhibits fibronectin-mediated adhesion. It also affects cell cycle progression of BCP-ALL cells. PTX008 is cytostatic for BP-ALL cells even when these are co-cultured with protective stroma, and can sensitize ALL cells to vincristine chemotherapy in vitro and in mice. CONCLUSIONS: PTX008 inhibits multiple functions that contribute to BP-ALL survival. The effects of Galectin-1 inhibition on both BP-ALL cell proliferation and migration suggest both the leukemia cells as well as the microenvironment that protects these cells may be targeted.
Assuntos
Antineoplásicos/farmacologia , Galectina 1/antagonistas & inibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Adesão Celular/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Modelos Animais de Doenças , Galectina 1/genética , Galectina 1/metabolismo , Expressão Gênica , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Ligação Proteica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The molecular interactions between B-cell precursor acute lymphoblastic leukemia (pre-B ALL) cells and stromal cells in the bone marrow that provide microenvironmentally-mediated protection against therapeutic drugs are not well-defined. Galectin-3 (Lgals3) is a multifunctional galactose-binding lectin with reported location in the nucleus, cytoplasm and extracellular space in different cell types. We previously reported that ALL cells co-cultured with stroma contain high levels of Galectin-3. We here establish that, in contrast to more mature B-lineage cancers, Galectin-3 detected in and on the ALL cells originates from stromal cells, which express it on their surface, secrete it as soluble protein and also in exosomes. Soluble and stromal-bound Galectin-3 is internalized by ALL cells, transported to the nucleus and stimulates transcription of endogenous LGALS3 mRNA. When human and mouse ALL cells develop tolerance to different drugs while in contact with protective stromal cells, Galectin-3 protein levels are consistently increased. This correlates with induction of Galectin-3 transcription in the ALL cells. Thus Galectin-3 sourced from stroma becomes supplemented by endogenous Galectin-3 production in the pre-B ALL cells that are under continuous stress from drug treatment. Our data suggest that stromal Galectin-3 may protect ALL cells through auto-induction of Galectin-3 mRNA and tonic NFκB pathway activation. Since endogenously synthesized Galectin-3 protects pre-B ALL cells against drug treatment, we identify Galectin-3 as one possible target to counteract the protective effects of stroma.
Assuntos
Galectina 3/metabolismo , Comunicação Parácrina , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Antineoplásicos/farmacologia , Proteínas Sanguíneas , Linhagem Celular Tumoral , Endocitose , Exossomos/metabolismo , Galectina 3/deficiência , Galectina 3/genética , Galectinas , Regulação Leucêmica da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/patologia , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , Microambiente TumoralRESUMO
Ginsenoside Rg3 (Rg3), a pharmacologically active compound from red ginseng, has been reported to induce cell death in various cancer cell lines, although the specific mechanisms have not been well established. In the present study, Rg3 treatment to A549 human lung adenocarcinoma led to cell death via not only apoptotic pathways but also the downregulation of epidermal growth factor receptor (EGFR). We used cross-linker and cell enzyme-linked immunosorbent assays to show that Rg3 inhibited EGFR dimerization by EGF stimulation and caused EGFR internalization from the cell membrane. Among several important phosphorylation sites in cytoplasmic EGFR, Rg3 increased the phosphorylation of tyrosine 1045 (pY1045) and serine 1046/1047 (pS1046/1047) for EGFR degradation and coincidently, attenuated pY1173 and pY1068 for mitogen-activated protein kinase activity. These effects were amplified under EGF-pretreated Rg3 stimulation. In vivo experiments showed that the average volume of the tumors treated with 30 mg/kg of Rg3 was significantly decreased by 40% compared with the control. Through immunohistochemistry, we detected the fragmentation of DNA, the accumulation of Rg3, and the reduction of EGFR expression in the Rg3-treated groups. Here, we provide the first description of the roles of Rg3 in the reduction of cell surface EGFR, the attenuation of EGFR signal transduction, and the eventual activation of apoptosis in A549 human lung adenocarcinoma.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Receptores ErbB/metabolismo , Ginsenosídeos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Receptores ErbB/análise , Ginsenosídeos/farmacologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos Endogâmicos C57BL , Panax/química , Multimerização Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacosRESUMO
Glycans play a critical role in physiological and pathological processes through interaction with a variety of ligands. Altered expression and dysregulation of these molecules can cause aberrant cellular function such as malignancy. Glycomics provide information of the structure and function of glycans, glycolipids, and glycoproteins such as proteoglycans, and may help to predict cancer development and progression as biomarkers. In this report, we compared the expression of proteoglycans, the content and structure of glycosaminoglycans and glycolipids between patient-matched normal and cancer tissues obtained from colon cancer patients. Tumor-related proteoglycans, glypican-3, and syndecan-1 showed downregulation in cancer tissues compared to normal tissues. In cancer tissue, the total amount of chondroitin sulfate (CS)/dermatan sulfate and heparan sulfate were lower and, interestingly, the level of disaccharide units of both 4S6S (CS-E) and 6S (CS-C) were higher compared to normal tissue. Also, overall lipids including glycolipids, a major glycomics target, were analyzed by hydrophilic interaction liquid chromatography mass spectrometry. Increase of lyso-phosphatidylcholine (phospholipid), sphingomyelin (sphigolipid), and four types of glycolipids (glucosylceramide, lactosylceramide, monosialic acid ganglioside, and globoside 4) in cancer tissue showed the possibility as potential biomarkers in colon cancer. While requiring the need for careful interpretation, this type of broad investigation gives us a better understanding of pathophysiological roles on glycosaminoglycans and glycolipids and might be a powerful tool for colon cancer diagnosis.
Assuntos
Adenocarcinoma/química , Neoplasias do Colo/química , Regulação Neoplásica da Expressão Gênica , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/análise , Antígenos CD/química , Biomarcadores/química , Sequência de Carboidratos , Estudos de Casos e Controles , Sulfatos de Condroitina/análise , Sulfatos de Condroitina/química , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/genética , Dermatan Sulfato/análogos & derivados , Dermatan Sulfato/análise , Dermatan Sulfato/química , Feminino , Gangliosídeos/análise , Gangliosídeos/química , Globosídeos/análise , Globosídeos/química , Glucosilceramidas/análise , Glucosilceramidas/química , Glipicanas/química , Glipicanas/genética , Heparitina Sulfato/análise , Heparitina Sulfato/química , Humanos , Lactosilceramidas/análise , Lactosilceramidas/química , Lisofosfatidilcolinas/análise , Lisofosfatidilcolinas/química , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Esfingomielinas/análise , Esfingomielinas/química , Sindecana-1/química , Sindecana-1/genéticaRESUMO
In this study, we investigated the anticancer mechanism of akebia saponin PA (AS), a natural product isolated from Dipsacus asperoides in human gastric cancer cell lines. It was shown that AS-induced cell death is caused by autophagy and apoptosis in AGS cells. The apoptosis-inducing effect of AS was characterized by annexin V/propidium (PI) staining, increase of sub-G1 phase and caspase-3 activation, while the autophagy-inducing effect was indicated by the formation of cytoplasmic vacuoles and microtubule-associated protein 1 light chain-3 II (LC3-II) conversion. The autophagy inhibitor bafilomycin A1 (BaF1) decreased AS-induced cell death and caspase-3 activation, but caspase-3 inhibitor Ac-DEVD-CHO did not affect LC3-II accumulation or AS-induced cell viability, suggesting that AS induces autophagic cell death and autophagy contributes to caspase-3-dependent apoptosis. Furthermore, AS activated p38/c-Jun N-terminal kinase (JNK), which could be inhibited by BaF1, and caspase-3 activation was attenuated by both SB202190 and SP600125, indicating that AS-induced autophagy promotes mitogen-activated protein kinases (MAPKs)-mediated apoptosis. Taken together, these results demonstrate that AS induces autophagic and apoptotic cell death and autophagy plays the main role in akebia saponin PA-induced cell death.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Saponinas/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Antineoplásicos Fitogênicos/antagonistas & inibidores , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Biomarcadores/metabolismo , Caspase 3/química , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dipsacaceae/química , Etnofarmacologia , Fase G1/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Medicina Tradicional Coreana , Estrutura Molecular , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Raízes de Plantas/química , Inibidores de Proteínas Quinases/farmacologia , República da Coreia , Saponinas/antagonistas & inibidores , Saponinas/química , Saponinas/isolamento & purificação , Neoplasias Gástricas/química , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
Glycosaminoglycans (GAGs) are complex carbohydrates that are ubiquitously present on the cell surface and in the extracellular matrix. Interactions between GAGs and pathogens represent the first line of contact between pathogen and host cell and are crucial to a pathogen's invasive potential. Their complexity and structural diversity allow GAGs to control a wide array of biological interactions influencing many physiological and pathological processes, including adhesion, cell-to-cell communication, biochemical cascades, and the immune response. In recent years, increasing evidence indicates an extraordinary role for GAGs in the pathogenesis of viruses, bacteria and parasites. Herein, we examine the interface between GAGs and different pathogens, and address the divergent biological functions of GAGs in infectious disease. We consider approaches to use this understanding to design novel therapeutic strategies addressing new challenges in the treatment of infectious diseases.
Assuntos
Doenças Transmissíveis/metabolismo , Glicosaminoglicanos/metabolismo , Animais , Comunicação Celular , Doenças Transmissíveis/imunologia , Regulação da Expressão Gênica , Glicosaminoglicanos/genética , Interações Hospedeiro-PatógenoRESUMO
Ginsenosides are active compounds isolated from Panax ginseng Meyer. Among these ginsenosides, less polar ginsenosides such as ginsenoside Rg3 and ginsenoside Rh2 have been demonstrated to have tumor inhibitory effects because of their cytotoxicity. In this study, we evaluated the apoptotic effects of ginsenoside Rk1 in SK-MEL-2 human melanoma. Ginsenoside Rk1 isolated from red ginseng is one of the novel ginsenosides that shows strong cytotoxicity compared to ginsenoside Rg3 in dose- and time-dependent manners. The results of DNA fragmentation, 4',6-diamidino-2-phenylindole staining, and flow cytometric analysis are corroborated that ginsenoside Rk1 induced apoptosis in SK-MEL-2 cells. Western blot analysis revealed up-regulation of Fas, FasL, and Bax protein expression and down-regulation of procaspase-8, procaspase-3, mutant p53 and Bcl-2 protein expression. These findings suggest that ginsenoside Rk1 might be a promising compound to induce apoptosis through both extrinsic and intrinsic pathways in SK-MEL-2 cells.